Cell separation and sorting techniques can be divided into two main categories: (1) methods that require tagging the cells with a label; (2) label-free methods. Flow cytometer is the gold standard for cell separation methods that rely on tagging the cells with labels. Flow cytometer is an instrument for measuring fluorescence and light scattering of individual microscopic particle/cells. Most commonly used flow cytometer techniques are fluorescent activated cell sorting (FACS) and magnetic activated cell sorting (MACS). FACS method utilizes complementary fluorophore-conjugated antibodies to label cells of interests. Although FACS offers high-throughput sorting, there are several technical drawbacks including equipment expense, clogging, contamination, deflection, and cell viability after ejection. Magnetic activated cell sorter (MACS) employs antibody-conjugated, magnetic beads to bind specific proteins on cells of interest. This method is capable of collecting many cells with a relatively low cost. There are, however, several disadvantages to the method including low sensitivity, sensitive process of labeling cells with the magnetic beads and long time frames are requirement for efficient separation. More importantly, both of these cytometer methods require additional “tags” or “labels” to identify cells. However, the biochemical markers may not be available for a specific population. Also, the use of labels may hinder differentiation and they may expand in vitro or in vivo and they add difficulty and cost to the procedure.
Therefore, various label-free cell separation methods have been investigated that address the limitations of flow cytometers. Pinched flow fractionation is a hydrodynamic chromatographic technique capable of separation only by size. As the separation efficiency is based solely on the laminar flow profile in the pinched and broadened segment, cells of varying sizes can be separated effectively by tuning the ratio of the sample to sheath buffer flow rates. However, due to dimension limitation of the pinched segment, separation of small (few microns) particles/cells is very challenging. Also, feasible separation efficiency can only be obtained with very limited flow rates, conflicting with high-throughput system efforts. Hydrodynamic filtration method is another effective way of cell separation, with the separation being determined purely by channel geometry effective. Although this method offers simple operation, the separation efficiency is too low to be a viable separation method. Deterministic lateral displacement technique sort cells exclusively by size using only the geometry of microchannel. Thus, this method does not require external force or field. However, there is a high risk of clogging due to the presence of a large number of high density post structures. Gravitational and sedimentation methods depend on the density, rather than the density of the medium. Separation is simple and does not impose any damaging mechanical stresses on the cells being separated. However, it is limited to the separation of cell populations with relatively large size difference, limiting its use. Optical lattice technique employs three-dimensional arrays of light traps, optical lattices, using holographic optical tweezers. This method has the advantages in term of sensitivity and selectivity. However, the need for laser source prevents the easy portability of the system and continuous system for processing large samples has not been demonstrated due to low separation efficiency. Throughput of this technique also is very limited when compared to flow cytometers. Dielectrophoresis method allows for distinguishing between live and dead cells as well as different types of bacteria by physical properties in addition to size. However, the throughputs of dielectrophoresis are still low when compared to other alternative. Moreover, the cells are often trapped improperly with positive dielectrophoresis and high electric fields could lead to Joule heating and bubble generation as well as heat-related cell death. As a result, none of these methods provides a generic solution to cell separation needs.